scholarly journals A nucleoside triphosphate-dependent deoxyribonucleic acid-breakdown system in Mycobacterium smegmatis, and the effect of iron limitation on the activity of this system

1969 ◽  
Vol 111 (5) ◽  
pp. 679-687 ◽  
Author(s):  
F. G. Winder ◽  
M. P. Coughlan
Keyword(s):  
Dna Content ◽  
Mycobacterium Smegmatis ◽  
Deoxyribonucleic Acid ◽  
Maximal Activity ◽  
Enzyme Concentration ◽  
Iron Limitation ◽  
Nucleoside Triphosphate ◽  
Primary Products ◽  
Rate Of Reaction ◽  
Optimum Ph

1. The presence of a nucleoside triphosphate-dependent DNA-breakdown system was demonstrated in extracts of Mycobacterium smegmatis. Its activity was increased substantially by iron limitation, apparently after the fall in DNA content that took place under these conditions. A maximal activity of about 0·2μmole of deoxyribonucleotide/30min./mg. of protein was found in crude extracts. 2. After slight purification by streptomycin treatment, the enzyme showed maximal activity with undenatured DNA (Km≃200μg./ml.), ATP (Km≃1·2mm) or UTP, CTP and GTP giving lower activity and pyrophosphate giving none, and Mg2+ ions (optimum concn. 12mm). The optimum pH was 8·5. 3. In the assay system there was proportionality between enzyme concentration and rate of reaction, but the rate fell off with time. 4. ATP was broken down in the reaction and monodeoxy-ribonucleotides were among the products, but the presence of some oligodeoxy-ribonucleotides was not excluded and the degree of phosphorylation of the primary products was uncertain.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

scholarly journals Evaluation of optimal conditions for arginase activity in streptozotocin induced diabetic rats

2012 ◽  
Vol 50 (No. 2) ◽  
pp. 69-76 ◽  
Author(s):  
M. Erisir ◽  
E. Ercel ◽  
S. Yilmaz ◽  
S. Ozan
Keyword(s):  
Maximal Activity ◽  
Diabetic Rats ◽  
Arginase Activity ◽  
Female Rats ◽  
Kinetic Properties ◽  
Ph Profile ◽  
Optimum Ph ◽  
Liver And Kidney ◽  
And Control ◽  
Assay Conditions

The assay conditions needed to achieve maximal activity of liver and kidney arginase in diabetic and non-diabetic rats were investigated and compared. The physicochemical and kinetic properties of liver arginase in diabetic and control rats were very similar, those of kidney arginase were significantly different. It was found that preincubation temperature (68&deg;C), preincubation period (20 min), optimum pH (10.1) of liver arginase and K<sub>m</sub> (3.2) for its substrate, L-arginine, did not change in diabetic and non-diabetic rats. As a consequence of diabetes, the optimum Mn<sup>2+</sup> concentration for liver arginase only changed from 1 to 2 mM. Although the preincubation temperature and period for activation of kidney arginase in control rats was unnecessary, they were found to be 56&ordm;C and 12 min in diabetic rats. The pH profile of arginase in kidney of diabetic rats was different from that of control rats. The K<sub>m</sub> value (6.7) of arginase for L-arginine in kidney is unchanged in diabetes whereas a marked decrease in V<sub>max</sub> was found. Optimum Mn<sup>2+</sup> concentration (2 mM) for kidney arginase was unchanged in diabetes. The activity of arginase in liver of diabetic animals was higher 1.5 to 1.7 times than that of controls. Diabetes caused an about 53% decrease of arginase activity in kidney of female rats, 26% in that of males. These findings may suggest an idea that encoded arginases by separate gene loci may be affected differently by the pathological and hormonal status.

scholarly journals Assays, Surrogates, and Alternative Technologies for a TB Lead Identification Program Targeting DNA Gyrase ATPase

2014 ◽  
Vol 20 (2) ◽  
pp. 265-274 ◽  
Author(s):  
Vaishali Humnabadkar ◽  
Prashanti Madhavapeddi ◽  
Halesha Basavarajappa ◽  
Md. Gulebahar Sheikh ◽  
Rajendra Rane ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Specific Activity ◽  
Dna Gyrase ◽  
Enzyme Concentration ◽  
Alternative Technologies ◽  
Lead Identification ◽  
Drug Discovery Program ◽  
Lead Generation ◽  
Nanomolar Range

Mycobacterium tuberculosis (Mtb) DNA gyrase ATPase was the target of a tuberculosis drug discovery program. The low specific activity of the Mtb ATPase prompted the use of Mycobacterium smegmatis (Msm) enzyme as a surrogate for lead generation, since it had 20-fold higher activity. Addition of GyrA or DNA did not significantly increase the activity of the Msm GyrB ATPase, and an assay was developed using GyrB alone. Inhibition of the Msm ATPase correlated well with inhibition of Mtb DNA gyrase supercoiling across three chemical scaffolds, justifying its use. As the IC50 of compounds approached the enzyme concentration, surrogate assays were used to estimate potencies (e.g., the shift in thermal melt of Mtb GyrB, which correlated well with IC50s >10 nM). Analysis using the Morrison equation enabled determination of [Formula: see text]s in the sub-nanomolar range. Surface plasmon resonance was used to confirm these IC50s and measure the Kds of binding, but a fragment of Mtb GyrB had to be used. Across three scaffolds, the dissociation half life, t1/2, of the inhibitor-target complex was ≤8 min. This toolkit of assays was developed to track the potency of enzyme inhibition and guide the chemistry for progression of compounds in a lead identification program.

THE HETEROGENEITY OF PANCREATIC DEOXYRIBONUCLEASE

1962 ◽  
Vol 40 (2) ◽  
pp. 165-175 ◽  
Author(s):  
G. C. Becking ◽  
R. O. Hurst
Keyword(s):  
Enzyme Activity ◽  
Ionic Strength ◽  
Enzyme Preparation ◽  
Deoxyribonucleic Acid ◽  
Paper Electrophoresis ◽  
Relative Activity ◽  
Enzyme Concentration ◽  
Uranyl Acetate ◽  
Ph Optimum

The action of crystalline pancreatic deoxyribonuclease on sodium oligonucleotides in the presence of manganous ions has been studied and a pH optimum of 6.6 observed. Inhibition of the enzyme activity by increased ionic strength of the digest occurred. The liberation of products soluble in uranyl acetate – trichloroacetate was found to vary with enzyme concentration and the relative activity of the enzyme on oligonucleotides was best determined by a logarithm-plot method. The activity of the enzyme towards deoxyribonucleic acid or sodium oligonucleotides as substrate was not affected by treatment with acetone. Evidence of heterogeneity in the crystalline enzyme preparation was obtained using paper electrophoresis and chromatography on carboxymethylcellulose. Two fractions were separated that showed different ratios of activity towards the two substrates employed.

Growth hormone and mammary gland lobule-alveolar development

1961 ◽  
Vol 201 (2) ◽  
pp. 259-263 ◽  
Author(s):  
Richard C. Moon
Keyword(s):  
Growth Hormone ◽  
Mammary Gland ◽  
Dna Content ◽  
Deoxyribonucleic Acid ◽  
Cellular Proliferation ◽  
Estradiol Benzoate ◽  
The Mean ◽  
Alveolar Formation ◽  
Inguinal Glands ◽  
Range Of Values

The effect of growth hormone on mammary gland lobule-alveolar growth in the ovariectomized rat was studied using deoxyribonucleic acid (DNA) of the abdominal-inguinal glands as an index of the degree of cellular proliferation. The administration of 1 mg growth hormone in combination with 2 µg estradiol benzoate for 19 days resulted in alveolar formation and an increase in mammary DNA content above that resulting from injections of either hormone alone. The mean DNA concentration of glands of rats treated with 2 µg estradiol, 6 mg progesterone, 3 µg/100 g l-thyroxine, and 0.5, 1.0, 1.5, and 2.0 mg growth hormone was significantly greater than that of animals receiving only the estradiol, progesterone, and thyroxine. The increase in the mean DNA content was due to a shift in the range of values to a higher plane and did not result from an elevated DNA in only a few animals. It is suggested that the administration of growth hormone during the growth phase of the mammary gland may have a beneficial effect on the subsequent lactation.

scholarly journals Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase

1971 ◽  
Vol 121 (4) ◽  
pp. 621-627 ◽  
Author(s):  
B. Gregory Louis ◽  
P. S. Fitt
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Rna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Halophilic Bacteria ◽  
Nucleoside Triphosphate ◽  
Extreme Halophile ◽  
Univalent Cations ◽  
Absolute Requirement ◽  
Nucleoside Triphosphates

1. DNA-dependent RNA polymerase was purified 150-fold from crude extracts of the extreme halophile Halobacterium cutirubrum. 2. The enzyme requires the presence of native DNA and all four nucleoside triphosphates to incorporate 14C-labelled nucleoside triphosphate into an acid-insoluble ribonuclease-sensitive product. 3. It has an absolute requirement for both Mn2+ and Mg2+. 4. The polymerase requires a high salt concentration for stability, but is markedly inhibited by univalent cations. 5. Its molecular weight is very low compared with that of Escherichia coli RNA polymerase.

HISTOCHEMICAL CHANGES IN THE SHOOT TIP OF CAULIFLOWER DURING FLORAL INDUCTION

1967 ◽  
Vol 45 (7) ◽  
pp. 955-959 ◽  
Author(s):  
Sidki Sadik ◽  
J. L. Ozbun
Keyword(s):  
Shoot Apex ◽  
Dna Content ◽  
Deoxyribonucleic Acid ◽  
Floral Induction ◽  
Fold Increase ◽  
Bromophenol Blue ◽  
Shoot Apices ◽  
Fast Green ◽  
Floral Primordia ◽  
Histochemical Changes

Cauliflower plants were induced to flower after being grown at 42 °F for varying periods of time, depending on the cultivar. Some of the histochemical changes in the shoot apex at the beginning of, during, and after floral induction were studied. During floral induction there is about a 20-fold increase in the volume of nucleoli and about a 3-fold increase in volume of nuclei. Apices of vegetative plants stained with bromophenol blue at pH 2.3, show small and dense nucleoli, dense and granular nuclei, and a small amount of weakly staining cytoplasm. In contrast, cells of apices of induced plants stained with bromophenol blue at pH 2.3, show large and dense nucleoli, large and weakly staining nuclei; however, these cells contain more and denser cytoplasm. Sections of vegetative and induced apices stained with alkaline fast green stained differently from those stained with bromophenol blue. Nucleoli did not stain and cytoplasm stained faintly with fast green while chromosomes stained strongly. Deoxyribonucleic acid (DNA) content of vegetative and induced apices are similar. Shoot apices of vegetative plants contained little or no starch. However, shoot apices of plants grown at 42 °F accumulate large amounts of starch. Floral primordia which develop into functional flowers are glutted with starch, while floral primordia which abort are void of starch.

scholarly journals MICROCHEMICAL DEOXYRIBONUCLEIC ACID DETERMINATION IN INDIVIDUAL CELLS

1961 ◽  
Vol 9 (3) ◽  
pp. 619-626 ◽  
Author(s):  
Jan-Erik Edström ◽  
Jerzy Kawiak
Keyword(s):  
Dna Content ◽  
Mitotic Cycle ◽  
Deoxyribonucleic Acid ◽  
Test Material ◽  
Cell Nuclei ◽  
Fixed Tissue ◽  
Dna Contents ◽  
Two Factors ◽  
Good Agreement

A method for the quantitative determination of DNA in the 50 to 500 µµg. range is presented. Cells or cell nuclei are isolated individually from fixed tissue by means of micromanipulation. The tissue units in question are extracted in an oil chamber with deoxyribonuclease solution. The extracts are evaporated to dryness and redissolved to lens-shaped drops, the DNA contents of which are determined by a photographic-photometric procedure in ultraviolet light. Determinations on calf thymocytes and rat spermatids show a relatively good agreement with biochemical data. The present method tends, however, to give some. what higher values than those reported earlier. The coefficient of variation for analytical values from test material is about ± 10 per cent. The method has been applied to cells from the axolotl, adults as well as tadpoles. Germ cells (spermatids and spermatocytes) do not show any evidence of a biological variation in DNA content. Cells from proliferating tissues give an increased spread of the DNA values. It could be shown, for epithelial cells, that there are at least two factors determining the DNA content of these cells. One is the fact that the cells are investigated at different phases of the mitotic cycle; the other is the fact that the DNA synthesis cycle occupies different ranges for different cells.

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